Drug for neuroprotection

ABSTRACT

A drug containing a calmodulin inhibitor as an active ingredient is disclosed. This drug is useful in the suppression of neuronal cell death, in particular brain neuronal cell death, due to, for example, cerebral ischemia. 
     Also, a drug containing a compound capable of inhibiting binding of calmodulin to a cytoskeltal protein as an active ingredient and a drug containing a compound suppressing the breakdown of a cytoskeltal protein as an active ingredient are disclosed. 
     These drugs are useful in the treatment and prevention of various diseases in the brain and sequelae thereof as well as in the prevention of relapses of these diseases.

FIELD OF THE INVENTION

This is a division of application Ser. No. 08/249,249 filed 25 May 1994,now U.S. Pat. No. 5,661,150.

This invention relates to a drug which is useful in the treatment andprevention of various diseases in the brain and sequelae thereof as wellas in the prevention of relapses of these diseases.

BACKGROUND OF THE INVENTION

Cerebrovascular disorders (the term "cerebrovascular disorders" as usedherein means disorders in various brain cells and intracerebral bloodvessels induced by, for example, ischemia) break out due to so-calledcerebral ischemia, wherein the blood stream in the brain is lowered tothe threshold or below, which is induced by occlusion of blood vesselcaused by a constriction of blood vessel, cerebral thrombosis orcerebral embolus. Kirino et al. Brain Res., 377:344-347 (1982)! foundout that when transient cerebral ischemia was loaded to a gerbil,so-called delayed neuronal cell death, i.e., slow and delayedhippocampal pyramidal neuronal cell death was observed 2 or 3 daysthereafter. Although attempts have been made to clarify the degenerativeprocess of neuron after central damage (for example, ischemia), thismechanism has not been clarified so far.

According to Siesjoe and Bengtsson J. Cereb. Blood Flow Metab., 9:127(1989)!, at ischemia, glutamate is liberated from the presynaptic sideto synaptic clefts and then binds to a glutamate receptor in thepostsynaptic side. Thus the calcium ion influx into the cells and theliberation of calcium ion from intracellular storage sites are promoted.At the same time, the extrusion of intracellular calcium ion issuppressed due to decreased calcium-ATPase activity. As a result, theintracellular calcium ion concentration is elevated, which results inthe neuronal cell death. Compared with the extracellular calcium ionconcentration, the intracellular calcium ion concentration is extremelylow. It is known that cells cannot survive when the intercellularcalcium ion concentration is elevated to a certain level. However theprocess from an increase in the intracellular calcium ion concentrationto neuronal cell death has not been clarified yet.

Also, there have been reported that at cerebral ischemia, calmodulin,which is a calcium-binding protein, was activated Picone et al., J.Cereb. Blood Flow Metab., 9:805-811 (1989)!, the activity ofcalmodulin-dependent protein kinase was changed Churn et al., Stroke,21:1715-1721 (1990)! and breakdown of fodrin, which is acalmodulin-binding cytoskeltal protein, was accelerated by cerebralischemia disorders Seubert et al., Brain Res., 492:366-370 (1989)!.

On the other hand, there have been also reported that calmodulinaccelerated the breakdown of fodrin by calpain Harris et al., J. Biol.Chem., 264:17401-17408 (1989)! and trifluoropelazine, which is acompound having a calmodulin-inhibition effect, suppressed the breakdownof fodrin Seubert et al., Synapse, 1:20-24 (1987)!. However it has neverbeen reported that these phenomena participate in neuronal cell death.

Although it is considered that a drug having a calmodulin-inhibitioneffect might be applicable to antihypertensive drug, antianginal drug,antiarrhythmic drug, drug for treating schizophrenia or drug forimproving cerebral circulation on the basis of vasodilator effect, noeffect of suppressing neuronal cell damage has been proved so far.Kogure et al. reported that as a result of their examination, W-7, whichis a substance having a calmodulin-inhibition effect, exhibited noeffect of suppressing delayed neuronal cell death and thus denied theapplicability of a calmodulin inhibitor as a drug for treatingcerebrovascular disorders Kogure et al., Tanpakushitsu, Kakusan, Koso,35:1254 (1990)!.

It has been reported that a phenothiazine compound, which also has aneffect of inhibiting calmodulin, relieved cerebral ischemic disorders byits antioxidant effect Ye et al., Stroke, 23:1287-1291 (1992)!.

With the tendency toward an aging society in recent years, increases inbrain disorders including cerebrovascular disorders and Alzheimer'sdisease have become a serious social problem. It is known that the rootof these diseases lies in brain neuronal cell death caused by variousfactors. For example, cerebrovascular disorders are induced by cerebralischemia and the severity of these diseases relates to the ischemicperiod. Slight ischemia brings about little problem, while prolongedischemia results in irreversible damages in the brain. Since maturedneurons are not regenerated through cell division any more, thesedisorders remain as permanent organic changes and strongly affect theprognosis.

Therefore, it is highly useful in treating cerebrovascular disorders andrelieving the sequelae thereof to suppress neuronal cell death. Further,it has been proposed that neuronal cell death based on the accelerateddegradation of cytoskeltal protein causes Alzheimer's disease.Accordingly, suppression of neuronal cell death is useful in thetreatment and prevention of Alzheimer's disease and relief of thesequelae thereof too.

If the excessive activation of calmodulin induced by an increase in theintracellular calcium ion concentration at neuronal cell disorders canbe suppressed by using a calmodulin inhibitor, therefore, neuronal celldeath can be suppressed.

Delayed neuronal cell death occurs several days after the break out ofcerebrovascular disorders. The present inventors have found out that thecontent of calmodulin in cytoplasm begins to decrease from the earlystage (more concretely, even 1 hour after ischemia) while the calmodulincontent in membrane fraction increases. This is the same phenomenon asthe one observed when an excessive amount of calcium ion is added to ahippocampal homogenate. It indicates that calcium ion binds tocalmodulin at cerebral ischemia and some part thereof translocates intothe membrane side. The present inventors have further clarified that thedegradation of fodrin, which is a cytoskeltal protein contained in themembrane, is accelerated by the addition of calcium ion and thatcalmodulin binds to the breakdown products.

The present inventors have further found out that in a cerebral ischemicmodel of gerbil, the breakdown of fodrin is accelerated prior toneuronal cell death. Thus they have examined compounds A and B, eachhaving a high selectivity for calmodulin and an intense calmodulininhibition effect, and consequently found out that these compoundssuppress the translocation of calmodulin into the membrane in the earlystage of cerebral ischemia, suppress the breakdown of fodrin and, in itsturn, suppress neuronal cell death. The present invention has been thuscompleted.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a drug for neuroprotectionwhich is characterized by containing a calmodulin inhibitor as an activeingredient.

Further, the present invention relates to a drug for suppressing braincell death which is characterized by containing a calmodulin inhibitoras an active ingredient.

Further, the present invention relates to a drug for suppressingneuronal cell death which is characterized by containing a calmodulininhibitor as an active ingredient.

Further, the present invention relates to a drug for suppressing brainneuronal cell death which is characterized by containing a calmodulininhibitor as an active ingredient.

Further, the present invention relates to a drug for neuroprotection atcerebral ischemia which is characterized by containing a calmodulininhibitor as an active ingredient.

Further, the present invention relates to a drug for suppressing braincell death at cerebral ischemia which is characterized by containing acalmodulin inhibitor as an active ingredient.

Further, the present invention relates to a drug for suppressingneuronal cell death at cerebral ischemia which is characterized bycontaining a calmodulin inhibitor as an active ingredient.

Further, the present invention relates to a drug for suppressing brainneuronal cell death at cerebral ischemia which is characterized bycontaining a calmodulin inhibitor as an active ingredient.

Further, the present invention relates to a drug for neuroprotectionwhich is characterized by containing a compound inhibiting binding ofcalmodulin to a cytoskeltal protein as an active ingredient.

Further, the present invention relates to a drug for suppressing braincell death which is characterized by containing a compound inhibitingbinding of calmodulin to a cytoskeltal protein as an active ingredient.

Further, the present invention relates to a drug for suppressingneuronal cell death which is characterized by containing a compoundinhibiting binding of calmodulin to a cytoskeltal protein as an activeingredient.

Further, the present invention relates to a drug for suppressing brainneuronal cell death which is characterized by containing a compoundinhibiting binding of calmodulin to a cytoskeltal protein as an activeingredient.

Further, the present invention relates to a drug for neuroprotection atcerebral ischemia which is characterized by containing a compoundinhibiting binding of calmodulin to a cytoskeltal protein as an activeingredient.

Further, the present invention relates to a drug for suppressing braincell death at cerebral ischemia which is characterized by containing acompound inhibiting binding of calmodulin to a cytoskeltal protein as anactive ingredient.

Further, the present invention relates to a drug for suppressingneuronal cell death at cerebral ischemia which is characterized bycontaining a compound inhibiting binding of calmodulin to a cytoskeltalprotein as an active ingredient.

Further, the present invention relates to a drug for suppressing brainneuronal cell death at cerebral ischemia which is characterized bycontaining a compound inhibiting binding of calmodulin to acytoskeltal-protein as an active ingredient.

Further, the present invention relates to a drug for neuroprotectionwhich is characterized by containing a compound suppressing thebreakdown of a cytoskeltal protein as an active ingredient.

Further, the present invention relates to a drug for suppressing braincell death which is characterized by containing a compound suppressingthe breakdown of a cytoskeltal protein as an active ingredient.

Further, the present invention relates to a drug for suppressingneuronal cell death which is characterized by containing a compoundsuppressing the breakdown of a cytoskeltal protein as an activeingredient.

Further, the present invention relates to a drug for suppressing brainneuronal cell death which is characterized by containing a compoundsuppressing the breakdown of a cytoskeltal protein as an activeingredient.

Further, the present invention relates to a drug for neuroprotection atcerebral ischemia which is characterized by containing a compoundsuppressing the breakdown of a cytoskeltal protein as an activeingredient.

Further, the present invention relates to a drug for suppressing braincell death at cerebral ischemia which is characterized by containing acompound suppressing the breakdown of a cytoskeltal protein as an activeingredient.

Further, the present invention relates to a drug for suppressingneuronal cell death at cerebral ischemia which is characterized bycontaining a compound suppressing the breakdown of a cytoskeltal proteinas an active ingredient.

Further, the present invention relates to a drug for suppressing brainneuronal cell death at cerebral ischemia which is characterized bycontaining a compound suppressing the breakdown of a cytoskeltal proteinas an active ingredient.

The term "a drug for neuroprotection" as used herein means a drug whichis to be used in order to suppress neuronal cell death to therebyprevent, relieve or treat various disorders in cerebral functions causedby this neuronal cell death.

DETAILED DESCRIPTION OF THE INVENTION

It has been considered that calmodulin inhibitors would be applicable todrugs for treating diseases in circulatory organs such asantihypertensive drug, antianginal drug, antiarrhythmic drug, and drugfor improving cerebral circulation or psychotropic drug. However, thefindings of the present inventors reveal that a calmodulin inhibitor isusable as a drug for treating cerebrovascular disorders. That is to say,a calmodulin inhibitor is highly useful as a drug for treating variousdiseases induced by the excessive activation of calmodulin. Inparticular, it is highly useful as a drug for treating or preventingcerebrovascular disorders (for example, cerebral infarction, cerebralembolus, transient cerebral ischemia, cerebral thrombosis), cerebraldenaturation diseases (for example, Alzheimer's disease, Parkinson'sdisease) and other cerebral disorders (for example, drug addiction, gaspoisoning, trauma cerebral diseases), and diseases induced thereby (forexample, depressed voluntariness, depression and disorders in memory).

As a matter of course, the present invention involves methods fortreating various diseases induced by the excessive activation ofcalmodulin which are characterized by administering a calmodulininhibitor.

The drug for neuroprotection of the present invention may beadministered either orally or parenterally.

The dose of the drug for neuroprotection of the present invention can beappropriately varied depending on the conditions, age, body weight andseverity of the patient. In the case of oral administration, it can beadministered to an adult in a dose of from 1 mg to 1000 mg, preferablyfrom 10 mg to 500 mg, per day. It may be administered either at once orin several portions. As examples of the administration form, tablets,capsules, dusts and granules may be cited. These preparations can beproduced by a publicly known method with the use of additives which arecommonly employed in the art, for example, excipients, lubricants andbinders.

In the case of parenteral administration, it can be administered to anadult in a dose of from 1 mg to 500 mg, preferably from 10 mg to 200 mg,per day. As preferable administration routes, subcutaneous intravenousinjection and intravenous drip infusion may be cited.

The drug for neuroprotection of the present invention can be formulatedinto a preparation by a widely known method. Now a formulation with theuse of the compound B employed in the experiments described herein willbe given by way of example.

    ______________________________________                                         Formulation Example 1!                                                       ______________________________________                                        (1) compound B       10        g                                              (2) lactose          50        g                                              (3) corn starch      15        g                                              (4) hydroxypropylcellulose                                                                         8         g                                              (5) carboxymethylstarch sodium                                                                     7         g                                              (6) magesium stearate                                                                              1         g                                              ______________________________________                                    

The above-mentioned components (1), (2), (3) and (5) are homogeneouslymixed in a fluidized bed granulating machine and granulated by using a6% aqueous solution of the component (4) as a binder. Then the component(5) is added thereto and homogeneously mixed to thereby give a powder tobe tabletted. Next, the powder was formulated into 100 tablets of 8 mmin diameter each containing 100 mg of the component (1).

    ______________________________________                                         Formulation Example 2!                                                       ______________________________________                                        (1) compound B      2          g                                              (2) 0.1 N hydrochloric acid                                                                       150        ml                                             (3) glucose         50         g                                              (4) distilled water for injection.                                            ______________________________________                                    

The above-mentioned components (1), (2) and (3) are mixed together andthe distilled water for injection was further added thereto so as toadjust the total volume to 1000 ml. The solution thus obtained wassterilely filtered through a 0.2 μm filter and pipetted in 10 mlportions into 10 ml ampuls.

It is expected that the drug for neuroprotection of the presentinvention would exert additional or multiplier effects of treating orpreventing various diseases when combined with other drugs. Examples ofsuch drugs include those for improving cerebral circulation (forexample, cinepazide maleate), drugs for improving cerebral metabolism(for example, idebenone, indeloxazine), psychotropics (for example,timiperone, imipramine, diazepam), drugs for lowering intracranialpressure (for example, glyceol), antihypertensive drugs, vasodilators(for example, trapidil), antipyretic analgesics, antiinflammatorysteroids, antiplatelets (for example, ticlopidine), anticoagulants (forexample, heparin), fibrinolytic drugs (for example, tissue plasminogenactivator), diuretics, antihyperlipemia (for example, probucol), drugsfor treating digestive ulcer, blood substituents, drugs for hepaticdiseases and drugs for malignant tumor.

To further illustrate the present invention in greater detail, and notby way of limitation, the following Examples will be given.

Pharmacological Example 1! Calmodulin inhibition effect

The calmodulin inhibition effect of a compound was evaluated by usingits effect of inhibiting calmodulin-depending phosphodiesterase (PDE) asan index. The experiment was performed by modifying the method ofThompson et al. Advances in Cyclic Nucleotide Research, 10, 69 (1979)!.Namely, 50 mM tris buffer (pH 7.5, containing 5 mM MgCl₂, and 1 mg/ml ofbovine serum albumin), 1 mM CaCl₂, ³ H!-cGMP, calmodulin (CaM, frombovine brain), CaM-PDE (calmodulin-depending phosphodiesterase, frombovine brain) and a specimen were mixed together and incubated at 30° C.for 10 minutes. After ceasing the reaction by heating in a boiling waterbath for 1 minute, snake venom (1 mg/ml) was added thereto and theresulting mixture was reacted at 30° C. for 10 minutes to therebyconvert 5'-GMP formed by PDE into guanosine. Next, the unreacted cGMPwas adsorbed by an ion exchange resin (AGI-X8). Subsequently, themixture was centrifuged and the radioactivity of the supernatant wasmeasured with a liquid scintillation counter. The inhibition effects(expressed in IC₅₀) of the employed compounds A and B thus determinedwere respectively 3.93 μM and 5.46 μM. On the other hand, the IC₅₀ ofW-7 employed as a control compound was 33.5 μM.

Pharmacological Example 2! Effect on intracellular localization ofhippocampal calmodulin

The hippocampus of a gerbil was homogenized with 20 mM tris buffer (pH7.5) containing 0.1 mM leupeptin, 0.1 mM PMSF and 0.01 mg/ml ofaprotinin. After adding a 1 mM aqueous solution of calcium chloride or 1mM EGTA ethylene-bis(oxyethylenenitrylo)tetraacetic acid, --CH₂ OCH₂ CH₂N-(CH₂ COOH)₂ !₂ !, it was incubated at 37° C. for 30 minutes. Thehomogenate was centrifuged at 100,000×g to thereby separate into asupernatant (a soluble fraction) and a precipitate (a membranefraction). The precipitate was solubilized with 0.1% Lubrol PX. Then thecalmodulin contents in the soluble fraction and the membrane fractionwere determined by radioimmunoassay. The calmodulin content in thesoluble fraction was significantly decreased by adding the aqueoussolution of calcium chloride to the hippocampal homogenate, comparedwith the one treated with EGTA. On the contrary, the content ofcalmodulin in the membrane fraction was increased by adding the aqueoussolution of calcium chloride. The compound A (10 μM) significantlysuppressed the change in calmodulin content caused by calcium ion. Table1 shows the results.

On the other hand, W-7 and the compound A exerted no effect on thebehavior of calmodulin in the presence of EGTA.

                  TABLE 1                                                         ______________________________________                                        Effect on intracellular localization                                          of calmodulin in hippocampus (1)                                                         Calmodulin Content (μg/mg protein)                              Test Group   Soluble Fraction                                                                            Membrane Fraction                                  ______________________________________                                        Control       4.5 ± 0.13                                                                               8.8 ± 0.25                                     1 mM EGTA     12.3 ± 0.77**                                                                            7.6 ± 0.40                                     1 mM CaCl.sub.2                                                                             0.83 ± 0.04**                                                                           11.4 ± 0.34**                                   1 mM CaCl.sub.2 +                                                                            1.75 ± 0.52**#                                                                          7.1 ± 0.50#                                    10 μM compound A                                                           ______________________________________                                         **: P < 0.01 vs. control. #: P < 0.05 vs. 1 mM CaCl.sub.2.               

Both common carotid arteries of a gerbil were ligated for 10 minutes.One hour and 24 hours after allowing the blood stream to flow again, thecalmodulin content in the hippocampus was determined byradioimmunoassay. The compound A (100 mg/kg) was suspended in 0.5%methylcellulose and orally administered to the animal 1 hour beforecerebral ischemia. The content of calmodulin in the cytoplasm (thesoluble fraction) was significantly decreased 1 hour after cerebralischemia, while the calmodulin content in the membrane fraction wasincreased on the contrary (Test 1). 24 hours after cerebral ischemia, anincrease in the calmodulin content was observed in the membranefraction. However, the compound A significantly suppressed such anincrease in the calmodulin content in the membrane fraction (Test 2).Table 2 shows the results.

                  TABLE 2                                                         ______________________________________                                        Effect on intracellular localization                                          of calmodulin in hippocampus (2)                                                          Calmodulin Content (μg/mg protein)                             Test Group    Cytoplasm Fraction                                                                         Membrane Fraction                                  ______________________________________                                        Test 1:                                                                       Normal        2.1 ± 0.12                                                                              2.1 ± 0.10                                      Cerebral ischemia                                                                           1.7 ± 0.08*                                                                             2.7 ± 0.22*                                     (after 1 hr)                                                                  Test 2:                                                                       Normal        1.0 ± 0.06                                                                              1.3 ± 0.10                                      Cerebral ischemia                                                                           1.1 ± 0.05                                                                              1.9 ± 0.10**                                    (after 24 hrs)                                                                Cerebral ischemia +                                                                         1.3 ± 0.08                                                                              1.4 ± 0.05                                      compound A                                                                    (100 mg/kg)                                                                   ______________________________________                                         *: P < 0.05. **: P < 0.01 vs. normal group. n = 10-12.                   

These pharmacological examples indicate the change in localization ofcalmodulin in the cells at the early stage of cerebral ischemia. Such achange was similar to the one induced by adding calcium ion to ahippocampal homogenate. Further, the compound A, which exerts a strongeffect of inhibiting calmodulin, suppressed the change in thelocalization of calmodulin in cells under the addition of calcium ion(in vitro) and in the cerebral ischemic model.

Pharmacological Example 3! Effect of calcium ion on breakdown ofcytoskeltal protein fodrin

The hippocampus taken out from a gerbil was homogenized with 20 mM trisbuffer (pH 7.5). After adding 1 mM CaCl₂ or 1 mM EGTA, it was incubatedat 37° C. for 1 hour. Then the homogenate was centrifuged at 10,000×gfor 30 minutes. After separating proteins contained in the supernatantfraction by SDS-PAGE, the proteins were identified by western blottingwith the use of a fodrin antibody (rabbit anti α-Spectrin) and acalmodulin antibody (sheep anti-bovine calmodulin). Under theEGTA-treatment, stable breakdown products of fodrin (140-150 kDa) werelittle observed. When Ca⁺⁺ was added, however, bands of the breakdownproducts appeared. As the result of the western blotting of calmodulin,bands assignable to calmodulin were observed almost at the samepositions of those assignable to fodrin and fodrin breakdown products,which indicates that calmodulin bound not only to fodrin but also tobreakdown products thereof.

Pharmacological Example 4! Change in fodrin in cerebral ischemic model

Both common carotid arteries of a gerbil were ligated for 10 minutes tothereby prepare a cerebral ischemic model. Four, 24 and 48 hours afterallowing the blood stream to flow again, the hippocampus was taken outand homogenized with 20 mM tris buffer (pH 7.5) containing 0.1 mMleupeptin, 0.1 mM PMSF and 0.15 mM aprotinin. After centrifuging at10,000×g for 30 minutes, proteins in the supernatant were separated bySDS-PAGE. Then fodrin and breakdown products thereof were identified bythe same method as the one described in Pharmacological Example 3. Thecompound B (100 mg/kg) was administered 1 hour before cerebral ischemiaand evaluated 48 hours thereafter. Fodrin breakdown products were littledetected from the normal gerbil. In the case of the cerebral ischemicmodel, on the other hand, breakdown products appeared 4 hours afterallowing the blood stream to flow again and similar bands were observedafter 48 hours. These fodrin breakdown products due to cerebral ischemiawere scarcely detected under the treatment with the compound B having astrong calmodulin inhibition effect.

Pharmacological Experiment 5! Change in hippocampal neuronal cells incerebral ischemic model

When transient cerebral ischemia was loaded to a gerbil, necrosis ofhippocampal cells was observed from several days thereafter. This changeis called delayed neuronal cell death. Cerebral ischemia was loaded to agerbil for 5 minutes. Seven days thereafter, the animal was sacrificedand the neuronal cells remaining in the hippocampal CA1 region werecounted. Most of the hippocampal CA1 cells died due to the cerebralischemia. When the compound A or B (100 mg/kg) was orally administeredto the animal 1 hour after cerebral ischemia, a protective effect onneuronal cells was clearly observed. Table 3 shows the results.

                  TABLE 3                                                         ______________________________________                                        Change in hippocampal neuronal                                                cells in cerebral ischemic model                                                                Hippocampal Neuronal Cell                                                     Density (/mm)                                               ______________________________________                                        Normal              194 ± 6.1                                              Cerebral ischemia    9 ± 1.3                                               Cerebral ischemia + compound A                                                                     98 ± 22.0**                                           Cerebral ischemia + compound B                                                                    131 ± 21.2**                                           ______________________________________                                         **: P < 0.01 vs. cerebral ischemia group. n = 9-10.                      

Referential Example 1! Ethyl5,6-dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazole-3-carboxylate

Ethyl 5,6-dimethoxy-1H-indazole-3-carboxylate (250.2 g) was suspended indimethyl sulfoxide (5000 ml, dried with Molecular Sieve 4A). Thenlithium methoxide (38.0 g) was added thereto and the mixture was stirredat room temperature. After stirring at room temperature for 1 hour,3,4-dimethoxybenzyl chloride (185.6 g) prepared from 336.4 g of3,4-dimethoxybenzyl alcohol, 300 ml of conc. hydrochloric acid and 500ml of diethyl ether! was added dropwise to the mixture at roomtemperature within 10 minutes. Then the mixture was stirred at roomtemperature for 1 hour. After adding 3,4-dimethoxybenzyl chloride (55.6g), the mixture was stirred at room temperature for 1 hour. Further,3,4-dimethoxybenzyl chloride (55.6 g) was added and the mixture wasstirred at room temperature for additional 1 hour. The reaction mixturewas poured into ice-water (30000 ml) with stirring. The supernatant wasremoved by decantation and a residue was dissolved in chloroform (10000ml), dried over sodium sulfate, filtered and evaporated under reducedpressure. The residue (497.0 g) thus obtained was separated and purifiedby using silica gel columns (chloroform/carbon tetrachloride/ethylacetate=5/5/1, silica gel 2 kg×9, followed by ethyl acetate/hexane=2/1,silica gel 2 kg×4). The obtained eluate was recrystallized from ethylacetate. Thus 205.0 g of ethyl5,6-dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazole-3-carboxylate (m.p.:138°-141° C.) was obtained in the form of prism crystals.

IR (KBr)cm⁻¹ : 1728, 1496, 1266, 1216, 1204, 1138, 1022.

¹ H-NMRδ (ppm, CDCl₃): 1.49 (3H, t, J=6.8 Hz), 3,78 (3H, s), 3.85 (6H,s), 3.95 (3H, s), 4.53 (2H, q, J=6.8 Hz), 5.58 (2H, s), 6.63 (1H, s),6.76 (1H, s), 6.80 (2H, s), 7.56 (1H, s).

Elemental analysis: Calcd. for C₂₁ H₂₄ N₂ 0₆ ; C, 62.99%; H, 6.04%; N,7.00%. Found: C, 62.83%; H, 5.99%; N, 6.93%.

Referential Example 2! 5,6-Dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazole-3-methanol

5,6-Dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazole-3-carboxylate (205.0g) was ground in a mortar and suspended in tetrahydrofuran (1500 ml) atroom temperature. Then sodium borohydride (96.8 g) was added thereto andthe mixture was stirred at room temperature. Methanol (300 ml) wasslowly added dropwise thereinto within 30 minutes. After the completionof the addition, the reaction mixture was heated to 50° C. and stirredfor 5 hours. After adding sodium borohydride (19.4 g) and methanol (60ml), the mixture was stirred at room temperature overnight. Then thereaction mixture was added in portions into a mixture of conc.hydrochloric acid (200 ml), water (5000 ml) and ice (1 kg) understirring (pH 1-2). To this aqueous layer, a saturated aqueous solutionof sodium bicarbonate was added with stirring at room temperature untilthe pH became about 8. As a result, a colorless solid was precipitated.This precipitate was collected by filtration, washed with water (500ml×2), dissolved in chloroform (10000 ml), dried over sodium sulfate,filtered and the solvent was evaporated under reduced pressure. Thus acolorless solid (185.2 g) was obtained. This solid product was used inthe subsequent reaction without purification.

Separately, a small amount of the solid product was taken andrecrystallized from ethanol to give colorless prism crystals (m.p.:187°-188° C.).

IR (KBr)cm⁻¹ : 3272, 1520, 1470, 1438, 1418, 1318, 1284, 1256, 1210,1166, 1140, 1062, 1026, 870, 834.

¹ H-NMRδ (ppm, CDCl₃): 3.77 (3H, s), 3.82 (3H, s), 3.87 (3H, s), 3.92(3H, s), 4.97 (2H, s), 5.40 (2H, s), 6.62 (1H, s), 6.69 (1H, m), 6.75(2H, m), 7.13 (1H, s).

Referential Example 3! 3-Chloromethyl-5,6-dimethoxy-1-(34-dimethoxybenzyl-1H-indazole

5,6-Dimethoxy-1-(3,4-dimethoxybenzyl)-3-hydroxymethyl-1H-indazole (184.0g) was dissolved in dichloromethane (1500 ml) at room temperature. Afterthe dissolution, the reaction mixture was ice-cooled and stirred. Thenthionyl chloride (75.4 ml) was dropped thereinto within 20 minutes. Thereaction mixture was stirred at room temperature and dichloromethane(3500 ml) was added thereto. Then it was washed with a saturated aqueoussolution of sodium bicarbonate (1000 ml), dried over sodium sulfate,filtered and the solvent was evaporated under reduced pressure. Thus189.7 g of a colorless solid was obtained. This solid product was usedin the subsequent reaction without purification.

¹ H-NMR8 (ppm, CDCl₃): 3.78 (3H, s), 3.84 (3H, s), 3.88 (3H, s), 3.95(3H, s), 4.95 (2H, s), 5.44 (2H, s), 6.65 (1H, s), 6.71 (3H, m), 7.10(1H, s).

Referential Example 4! 5,6-Dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazole-3-acetonitrile

3-Chloromethyl-5,6-dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazole (187.0g) was dissolved in dimethyl sulfoxide (1000 ml) and a solution wasstirred at room temperature. Then sodium cyanide (134.0 g) which hadbeen ground in a mortar was added thereto. The reaction mixture wasstirred at 50° C. for 2 hours. The reaction mixture was returned to roomtemperature, poured into water (15000 ml) and stirred for 1 hour. Thesolid thus precipitated was collected by filtration, washed with water(1000 ml×3), dissolved in chloroform (5000 ml), dried over sodiumsulfate, filtered and evaporated under reduced pressure. The residuethus obtained was purified by using silica gel columns(chloroform/ethanol 50/1, silica gel 2 kg, followed by silica gel 2 kg,ethyl acetate/hexane=3:1) to give 111.0 g of a pale brown solid product.This solid product was used in the subsequent reaction withoutpurification.

¹ H-NMRδ (ppm, CDCl₃): 3.80 (3H, s), 3.84 (3H, s), 3.89 (3H, s), 3.94(3H, s), 4.02 (2H, s), 5.43 (2H, s), 6.66 (1H, s), 6.72 (2H, m), 6.69(1H, m), 7.06 (1H, m).

Referential Example 5!5,6-Dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazole-3-acetic acid

5,6-Dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazole-3-acetonitrile (111.0g) was suspended in ethanol (1000 ml) at room temperature with stirring.Then a 10N aqueous solution of sodium hydroxide was added thereto andthe mixture was heated under reflux for 2 hours. The reaction mixturewas returned to room temperature and the ethanol (about 1000 ml) wasevaporated under reduced pressure. Then water (2000 ml) was addedthereto and the mixture was stirred overnight. After filtering off theinsoluble matters, ether (500 ml) was added and materials soluble in theorganic solvent was removed by discarding the organic layer. The aqueouslayer was adjusted to pH 4 to 5 by adding conc. hydrochloric acid. Thusa solid was precipitated. This precipitate was collected by filtrationand fractionally recrystallized from ethanol. Thus 41.0 g of5,6-dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazole-3-acetic acid wasobtained. This product was used in the subsequent reaction withoutfurther purification.

¹ H-NMR8 (ppm, CDCl₃): 3.77 (3H, s), 3.84 (3H, s), 3.88 (3H, s), 3.91(3H, s), 4.03 (2H, s), 5.44 (2H, s), 6.64 (1H, s), 6.72 (2H, m), 6.77(1H, m), 6.96 (1H, s).

Referential Example 6!1-((5,6-dimethoxy-1-(3,4-dimethoxybenzyl-1H-indazol-3-yl)acetyl)-4-(3-chloro-2-methylphenyl)piperazine

5,6-dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazole-3-acetic acid (41.0 g)was suspended in dichloromethane (500 ml). Then 2,2-dipyridyl disulfide(24.5 g) and triphenylphosphine (30.0 g) were added thereto and themixture was stirred at room temperature. Next,(3-chloro-2-methylphenyl)piperazine (23.5 g) dissolved indichloromethane (200 ml) was dropped thereinto within 5 minutes and themixture was stirred at room temperature for 30 minutes. Thendichloromethane (1000 ml) was added to the reaction mixture. Afterwashing with water, the organic layer was dried over sodium sulfate,filtered and evaporated under reduced pressure. The residue thusobtained was purified by using a silica gel column (ethylacetate/hexane=2/1, silica gel 2 kg) to give 61.5 g of a colorlesssolid. This solid product was used in the subsequent reaction withoutpurification. A small amount of this product was taken andrecrystallized from ethanol. Thus colorless prism crystals (m.p.:165°-169° C.) were obtained.

IR(KBr)cm⁻¹ : 1652, 1516, 1264, 1236.

¹ H-NMRδ (ppm, CDCl₃): 1.24 (1.5H, t, J=7.3 Hz, Me of EtOH), 1.65 (4H,s), 2.55 (2H, m), 2.75 (2H, m), 3.72 (1H, m, CH₂ of EtOH), 3.76 (3H, s),3.78 (3H, s), 3.89 (3H, s), 3.94 (3H, s), 4.09 (2H, s), 5.41 (2H, s),6.65 (1H, s), 6.69 (2H, m), 6.73 (1H, s), 7.03 (1H, t, J=7.8 Hz), 7.09(1H, d, J=6.8 Hz), 7.19 (1H, s).

Referential Example 7! 3-(2-(4-(3-chloro-2-methylphenyl)-1-piperazinylethyl)-5,6-dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazole (compound A)

1-((5,6-Dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazol-3-yl)acetyl)-4-(3-chloro-2-methylphenyl)piperazine(60.5 g) was suspended in tetrahydrofuran (1000 ml). Then 1.0 mol-boranetetrahydrofuran complex tetrahydrofuran solution (500 ml) was addedthereto and the mixture was heated under reflux for 2 hours. Thereaction mixture was cooled to room temperature and water (30 ml) wasadded to thereby decompose the excessive reagent. After evaporation ofthe tetrahydrofuran under reduced pressure, conc. hydrochloric acid (300ml) was added and the mixture was stirred at 50° C. for 1 hour. Theaqueous layer was returned to room temperature and made alkaline withpotassium carbonate. Then it was extracted with chloroform (3000 ml) andthe organic layer was dried over sodium sulfate, filtered and evaporatedunder reduced pressure. The residue thus obtained was purified by usinga silica gel column (chloroform/ethanol=40/1). Thus a colorless solid(50.0 g) was obtained. This product was recrystallized from ethanol togive 46.3 g of colorless prism crystals (m.p.: 148°-150° C.)

IR(KBr)cm⁻¹ : 1518, 1466, 1454, 1260, 1236, 1140, 1022, 1004.

¹ H-NMRδ (ppm, CDCl₃): 2.35 (3H, s), 2.85 (2H, m), 3.02 (4H, m), 3.26(2H, m), 3.78 (3H, s), 3.83 (3H, s), 3,87 (3H, s), 3.94 (3H, s), 5.43(2H, s), 6.62 (1H, s), 6.72 (2H, s), 6.78 (1H, m), 6.96 (1H, m), 7.11(3H, m).

Elemental analysis: calcd. for C₃₁ H₃₇ N₄ O₄ Cl: C, 65.89%; H, 6.60%; N,9.91%; Cl, 6.27% found: C, 65.65%; H, 6.59%; N, 9.58%; Cl, 6.36%.

Referential Example 8!5,6-Dimethoxy-1-(1-trityl-4-imidazolyl)methyl-1H-indazole-3-methanol

Ethyl5,6-dimethoxy-1-(1-trityl-4-imidazolyl)methyl)-1H-indazole-3-carboxylate(222.0 g), which had been ground in a mortar, was suspended intetrahydrofuran (1300 ml) at room temperature and cooled with ice/water.Next, sodium bis-methoxyethoxy aluminum hydride (3.4M toluene solution,ca. 250.0 ml) was added thereto within 15 minutes and cooled withice/water under stirringm for 30 minutes. A persaturated aqueoussolution of sodium sulfate was added to the reaction mixture and stirredfor 1 hour. Then sodium sulfate was added thereto and the mixture wasfiltered. The sodium sulfate on the filter was washed with hotchloroform (500 ml×5). After concentrating the filtrate, a colorlesssolid (220.1 g) was obtained. This product was recrystallized fromchloroform to give 181.0 g of colorless prism crystals m.p.: 115°-120°C. (dec.)!.

IR(KBr)cm⁻¹ : 3216, 3172, 3008, 2936, 1510, 1488, 1472, 1444,1302, 1260,1172, 1156, 1128, 1102, 1036, 1014, 836, 764, 746, 702, 678, 666, 636.

¹ H-NMRδ (ppm, CDCl₃): 3.91 (3H, s), 3.92 (3H, s), 4.92 (2H, s), 5.44(2H, s), 6.76 (1H, s), 6.95 (1H, s), 7.05 (5H, m), 7.26 (1H, s, CHCl₃),7.28 (1H, s), 7.31 (10H, m), 7.46 (1H, s).

Elemental analysis: Calcd. for C₃₃ H₃₀ N₄ O₃.CHCl₃ : C, 62.83; H, 4.81;N, 8.62. Found: C, 62.50; H, 4.63; N, 8.42.

Referential Example 9!3-Chloromethyl-5,6-dimethoxy-1-(1-trityl-4-imidazolyl methyl-1H-indazole

5,6-Dimethoxy-1-(1-trityl-4-imidazolyl)methyl-1H-indazole-3-methanol(180.0 g), which had been ground in a mortar, was suspended indichloromethane (1700 ml) at room temperature and then cooled withice/water. Next, thionyl chloride (48.6 ml) was dropped thereinto within5 minutes. The reaction mixture was poured into a saturated aqueoussolution of sodium bicarbonate (2000 ml) and extracted with chloroform(5000 ml). After drying over sodium sulfate, filtering and evaporatingunder reduced pressure, a colorless solid was obtained (165.1 g). Thissolid product was used in the subsequent reaction without purification.

¹ H-NMRδ (ppm, CDCl₃): 3.95 (3H, s), 4.09 (3H, s), 4.83 (2H, s), 5.67(2H, s), 7.02 (8H, m), 7.37 (10H, m),. 7.88 (1H, br).

Referential Example 10! 5,6-Dimethoxy-1-(1-trityl-4-imidazolylmethyl-1H-indazole-3-acetonitrile

3-Chloromethyl-5,6-dimethoxy-1-(1-trityl-4-imidazolyl)methyl-1H-indazole(165.0 g) was suspended in dimethyl sulfoxide (1200 ml) and the solutionwas stirred at room temperature. Then potassium cyanide (43.6 g), whichhad been ground in a mortar, was added thereto. After stirring at 70° C.for 1 hour, the reaction mixture became homogeneous and transparent. Thereaction mixture was returned to room temperature and poured into water(15000 ml) while vigorously stirring. The stirring was continued for 1hour. The solid thus precipitated was collected by filtration, washedwith water (1000 ml×3), dissolved in chloroform (5000 ml), dried oversodium sulfate, filtered and evaporated under reduced pressure. Theresidue thus obtained was purified by using a silica gel column (ethylacetate) to give 108.7 g of a pale brown solid. This solid product wasused in the subsequent reaction without purification.

¹ H-NMRδ (ppm, CDCl₃): 3.92 (3H, s), 3.94 (3H, s), 3.97 (2H, s), 5.42(2H, s), 6.79 (1H, s), 7.00 (1H, s), 7.02 (1H, s), 7.06 (5H, m), 7.30(10H, m), 7.46 (1H, s).

Referential Example 11! 5,6-Dimethoxy-1-(1-trityl-4-imidazolylmethyl-1H-indazole-3-acetic acid

5,6-Dimethoxy-1-(1-trityl-4-imidazolyl)methyl-1H-indazole-3-acetonitrile(107.0 g) was suspended in ethanol (1000 ml) at room temperature. Then a10N aqueous solution of sodium hydroxide (prepared from 40.0 g of sodiumhydroxide and 100 ml of water) was added thereto and the mixture washeated under reflux for 6 hours. The reaction mixture was returned toroom temperature and poured into water (5000 ml). Then it was adjustedto pH 3 to 4 with a 10% aqueous solution of hydrochloric acid. As aresult, a colorless solid was precipitated. Then it was filtered andwashed with water (500 ml×3). The solid product thus obtained wasdissolved in chloroform (5000 ml), dried over sodium sulfate, filteredand evaporated under reduced pressure. The solid product (134.0 g) thusobtained was used in the subsequent reaction without purification.

¹ H-NMRδ (ppm, CDCl₃): 3.84 (3H, s), 3.87 (3H, s), 3.89 (2H, s), 5.43(2H, s), 6.76 (1H, s), 6.88 (1H, s), 6.93 (1H, s), 7.03 (5H, m), 7.28(10H, m), 7.48 (1H, s).

Referential Example 12!4-(3-Chloro-2-methylphenyl)-1-((5,6-dimethoxy-1-(1-trityl-4-imidazolyl)methyl-1H-indazol-3-yl)acetyl)piperazine

5,6-Dimethoxy-1-(1-trityl-4-imidazolyl)methyl-1H-indazole-3-acetic acid(134.0 g) was suspended in dichloromethane (1000 ml). Then 2,2-dipyridyldisulfide (63.5 g) and triphenylphosphine (75.6 g) were added theretoand the mixture was stirred at room temperature (the suspension becametransparent and homogeneous). Next, a solution of4-(3-chloro-2-methylphenyl)piperazine (60.7 g) in dichloromethane (200ml) was dropped thereinto within 5 minutes and the mixture was stirredat room temperature for 5 hours. The dichloromethane was evaporated fromthe reaction mixture under reduced pressure. To the residue, hot ethylacetate was added and stirred. As a result, a solid was precipitated.Then it was colleated by filtration, washed with ethyl acetate (500ml×2) and air-dried to give 140.4 g of a colorless solid. This solidproduct was purified by using a silica gel column(chloroform/ethanol=30/1) to give 134.9 g of a colorless solid. Next, itwas recrystallized from ethanol to give 120.0 g of colorless prismcrystals (m.p.: 103°-105° C.).

IR(KBr)cm⁻¹ : 1646, 1628, 1508, 1466, 1450, 1430, 1260, 750, 702.

¹ H-NMRS (ppm, CDCl₃): 1.23 (1.2H, t, J=6.8 Hz, Me of EtOH), 2.28 (3H,s), 2.55 (2H, m), 2.73 (2H, m), 3.67 (4H, m), 3.71 (0.8H, q, J=6.8 Hz,CH₂ of EtOH), 3.90 (3H, s), 3.93 (3H, s), 4.03 (2H, s), 5.43 (2H, s),6.68 (1H, s), 6.72 (1H, d, J=8.3 Hz), 6.90 (1H, s), 7.03 (7H, m), 7.14(1H, s), 7.27 (10H, m), 7.41 (1H, s).

Elemental analysis: calcd. for C₄₅ H₄₃ N₆ O₃ Cl.0.4EtOH H₂ O: C, 70.10%;H, 5.70%; N, 10.70%; Cl, 4.72%. found: C, 70.02%; H, 5.78%; N, 10.60%;Cl, 5.11%.

Referential Example 13!3-(2-(4-(3-Chloro-2-methylphenyl)-1-piperazinyl)ethyl-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole(compound B)

4-(3-chloro-2-methylphenyl)-1-((5,6-dimethoxy-1-(1-trityl-4-imidazolyl)methyl)indazol-3-yl)acetyl)piperazine(120.0 g) was suspended in tetrahydrofuran (1000 ml). Then 1.0M-boranetetrahydrofuran complex (800 ml) was added thereto and the mixture washeated under reflux for 90 minutes. The reaction mixture was cooled toroom temperature and water (30 ml) was added to decompose the excessivereagent. After evaporating the tetrahydrofuran under reduced pressure,conc. hydrochloric acid (150 ml), water (200 ml) and ethanol (40 ml)were added and the mixture was stirred at 50° C. for 1 hour. The aqueouslayer was stirred at room temperature and extracted with chloroform(3000 ml) which had been made alkaline with potassium carbonate. Theorganic layer was dried over sodium sulfate, filtered and distilled offunder reduced pressure. The residue thus obtained was purified by silicagel column chromatography (chloroform/ethanol=40/1) to give a colorlesssolid product. It was recrystallized from isopropyl alcohol-isopropylether and thus 71.0 g of colorless prism crystals (m.p.: 143°-144.5° C.)were obtained.

IR(KBr)cm⁻¹ : 1510, 1464, 1432, 1272, 1238, 1206, 1006.

¹ H-NMRS (ppm, CDCl₃): 2.34 (3H, s), 2.78 (4H, m), 2.90 (2H, m), 2.97(4H, m), 3.17 (2H, m), 3.90 (3H, s), 3.91 (3H, s), 5.45 (2H, s), 6.83(1H, s), 6.84 (1H, s), 6.92 (1H, m), 7.00 (1H, s), 7.09 (2H, m), 7.52(1H, s).

Elemental analysis: calcd. for C₂₆ H₃₁ N₆ 0₂ Cl: C, 63.09%; H, 6.31%; N,16.98%; Cl, 7.16%. found: C, 62.93%; H, 6.30%; N, 16.88%; Cl, 7.16%.

The present inventors have clarified: 1) intra-cellular localization ofcalmodulin is changed by an increase in calcium ion concentration due tocerebral ischemia or addition of calcium ion; 2) activated calmodulin(calmodulin binding to calcium ion) translocates toward cell membraneand binds to fodrin, which is a protein lining the cell membrane, tothereby accelerate the breakdown of fodrin; and 3) the test compounds Aand B, having strong effects compared with the existing calmodulininhibitors, suppress neuronal cell death. That is to say, they haveclarified that the activation of calmodulin and acceleration ofbreakdown of fodrin play important roles in neuronal cell death causedby cerebral ischemia. Therefore, a chemical capable of suppressing theabnormal activation of calmodulin is useful as a drug for neuronaldamage.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A method of neuroprotection comprisingadministering to a subject in need of treatment a pharmaceuticalcomposition comprising a compound which inhibits binding of calmodulinto a cytoskeletal protein in a neuroprotective amount and apharmaceutically acceptable carrier, excipient or diluent.
 2. A methodof neuroprotection at cerebral ischemia comprising administering to asubject with cerebral ischemia a pharmaceutical composition comprising acompound which inhibits binding of calmodulin to a cytoskeletal proteinsin a neuroprotective amount at cerebral ischemia and a pharmaceuticallyacceptable carrier, excipient or diluent.
 3. A method of neuroprotectioncomprising administering to a subject in need of treatment apharmaceutical composition comprising a compound which suppresses thebreakdown of a cytoskeletal protein in a neuroprotective amount atcerebral ischemia and a pharmaceutically acceptable carrier, excipientor diluent.
 4. A method of neuroprotection at cerebral ischemiacomprising administering to a subject with cerebral ischemia apharmaceutical composition comprising a compound which suppresses thebreakdown of a cytoskeletal protein in a neuroprotective amount atcerebral ischemia and a pharmaceutically acceptable carrier, excipientor diluent.
 5. A method of suppressing brain cell death comprisingexposing brain cells to a pharmaceutical composition comprising acompound which inhibits binding of calmodulin to a cytoskeletal proteinin an amount which suppresses brain cell death and a pharmaceuticallyacceptable carrier, excipient or diluent.
 6. A method of suppressingneuronal cell death comprising exposing neuronal cells to apharmaceutical composition comprising a compound which inhibits bindingof calmodulin to a cytoskeletal protein in an amount which suppressesneuronal cell death and a pharmaceutically acceptable carrier, excipientor diluent.
 7. A method of suppressing brain neuronal cell deathcomprising exposing brain neuronal cells to a pharmaceutical compositioncomprising a compound which inhibits binding of calmodulin to acytoskeletal protein in an amount which suppresses brain neuronal celldeath and a pharmaceutically acceptable carrier, excipient or diluent.8. A method of suppressing brain cell death at cerebral ischemiacomprising administering to a subject with cerebral ischemia apharmaceutical composition comprising a compound which inhibits bindingof calmodulin to a cytoskeletal protein in an amount which suppressesbrain cell death at cerebral ischemia and a pharmaceutically acceptablecarrier, excipient or diluent.
 9. A method of suppressing neuronal celldeath at cerebral ischemia comprising administering to a subject withcerebral ischemia a pharmaceutical composition comprising a compoundwhich inhibits binding of calmodulin to a cytoskeletal protein in anamount which suppresses neuronal cell death at cerebral ischemia and apharmaceutically acceptable carrier, excipient or diluent.
 10. A methodof suppressing brain neuronal cell death at cerebral ischemia comprisingadministering to a subject with cerebral ischemia a pharmaceuticalcomposition comprising a compound which inhibits binding of calmodulinto a cytoskeletal protein in an amount which suppresses brain neuronalcell death at cerebral ischemia and a pharmaceutically acceptablecarrier, excipient or diluent.
 11. A method of suppressing brain celldeath comprising exposing brain cells to a pharmaceutical compositioncomprising a compound which suppresses the breakdown of a cytoskeletalprotein in an amount which suppresses brain cell death and apharmaceutically acceptable carrier, excipient or diluent.
 12. A methodof suppressing neuronal cell death comprising exposing neuronal cells toa pharmaceutical composition comprising a compound which suppresses thebreakdown of a cytoskeletal protein in an amount which suppressesneuronal cell death and a pharmaceutically acceptable carrier, excipientor diluent.
 13. A method of suppressing brain neuronal cell deathcomprising exposing brain neuronal cells to a pharmaceutical compositioncomprising a compound which suppresses the breakdown of a cytoskeletalprotein in an amount which suppresses brain neuronal cell death and apharmaceutically acceptable carrier, excipient or diluent.
 14. A methodof suppressing brain cell death at cerebral ischemia comprisingadministering to a subject with cerebral ischemia a pharmaceuticalcomposition comprising a compound which suppresses the breakdown of acytoskeletal protein in an amount which suppresses brain cell death atcerebral ischemia and a pharmaceutically acceptable carrier, excipientor diluent.
 15. A method of suppressing neuronal cell death at cerebralischemia comprising administering to a subject with cerebral ischemia apharmaceutical composition comprising a compound which suppresses thebreakdown of a cytoskeletal protein in an amount which suppressesneuronal cell death at cerebral ischemia and a pharmaceuticallyacceptable carrier, excipient or diluent.
 16. A method of suppressingbrain neuronal cell death at cerebral ischemia comprising administeringto a subject with cerebral ischemia a pharmaceutical compositioncomprising a compound which suppresses the breakdown of cytoskeletalprotein in an amount which suppresses brain neuronal cell death atcerebral ischemia and a pharmaceutically acceptable carrier, excipientor diluent.
 17. The method of any one of claims 1, 2, 5, 6, 7, 8, 9 or10, wherein said compound which inhibits binding of calmodulin to acytoskeletal protein in3-(2-(4-(3-chloro-2-methylphenyl)-1-piperazinyl)ethyl)-5,6-dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazoleor3-(2-(4-(3-chloro-2-methylphenyl)-1-piperazinyl)ethyl)-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole.18. The method of any one of claims 3, 4, 11, 12, 13, 14, 15 or 16,wherein said compound which suppresses the breakdown of a cytoskeletalprotein is 3-(2-(4-(3-chloro-2-methylphenyl)-1-piperazinyl)ethyl)-5,6-dimethoxy-1-(3,4-dimethoxybenzyl)-1H-indazole or3-(2-(4-(3-chloro-2-methylphenyl)-1-piperazinyl)ethyl)-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole.